Hyaluronidase 1 deficiency decreases bone mineral density in mice

Mucopolysaccharidosis IX is a lysosomal storage disorder caused by a deficiency in HYAL1, an enzyme that degrades hyaluronic acid at acidic pH. This disease causes juvenile arthritis in humans and osteoarthritis in the Hyal1 knockout mouse model. Our past research revealed that HYAL1 is strikingly upregulated (~ 25x) upon differentiation of bone marrow monocytes into osteoclasts. To investigate whether HYAL1 is involved in the differentiation and/or resorption activity of osteoclasts, and in bone remodeling in general, we analyzed several bone parameters in Hyal1 −/− mice and studied the differentiation and activity of their osteoclasts and osteoblasts when differentiated in vitro. These experiments revealed that, upon aging, HYAL1 deficient mice exhibit reduced femur length and a ~ 15% decrease in bone mineral density compared to wild-type mice. We found elevated osteoclast numbers in the femurs of these mice as well as an increase of the bone resorbing activity of Hyal1 −/− osteoclasts. Moreover, we detected decreased mineralization by Hyal1 −/− osteoblasts. Taken together with the observed accumulation of hyaluronic acid in Hyal1 −/− bones, these results support the premise that the catabolism of hyaluronic acid by osteoclasts and osteoblasts is an intrinsic part of bone remodeling.


Results
Hyal1 −/− osteoclasts differentiated in vitro exhibit increased resorption activity. We isolated monocytes from the bone marrow of 7-month-old wild-type (WT) and Hyal1 −/− mice 4,9 and cultured them in vitro for 5 days in the presence of M-CSF (Macrophage-Colony Stimulating Factor) to stimulate their proliferation, survival and differentiation into macrophages 13 . Then, RANKL (Receptor Activator of Nuclear factor Kappa-B Ligand) was added to induce osteoclastogenesis 14 . After 7 days of culture on a flat glass surface, the number and size of TRAP-positive multinucleated cells (with 3 or more nuclei) were assessed. No difference was found between WT and Hyal1 −/− groups (Fig. 1a-c), indicating that the knockout of Hyal1 does not slow down nor accelerate the differentiation process of osteoclasts in vitro.
Next, bone marrow-derived monocytes were differentiated into osteoclasts on thin bone slices obtained from bovine nasal bone to assess their polarization and bone matrix resorption activity, as described in 15 . Staining of the nuclei with Hoechst 33,258 and incubation with Alexa 488-phalloidin (to label the actin rings that delimit resorption lacunae) pointed out that the number of differentiated and polarized osteoclasts is similar, irrespective of the WT or Hyal1 −/− background (Fig. 2a,b). However, the number of actin rings was found increased (Fig. 2c, p < 0.05). Moreover, scanning electron microscopy analysis of resorbed bone area, as well an assay of the C-terminal Telopeptide of type I-collagen degradation products (CTX, i.e. bone degradation products) released in the culture medium, revealed that the bone resorbing activity of Hyal1 −/− osteoclasts is increased relative to WT osteoclasts ( Fig. 2d-f). Indeed, the percentage of resorbed area represented 22 ± 4% for WT cells, compared to 30 ± 3% for KO osteoclasts (Fig. 2d,e, p < 0.01). Concomitantly, the latter released more CTX fragments than the former (2.5-fold increase, Fig. 2h, p < 0.05). In addition, the number and size of resorption pits were found elevated for Hyal1 −/− osteoclasts (Fig. 2f,g, p < 0.05).

HYAL1 deficiency causes HA accumulation in mouse femurs.
To investigate whether the inactivation of Hyal1 alters the level of HA in bone, we stained HA using HABP (HA-binding protein) on femur sections of 18-month-old mice. This analysis showed a significant increase of the HA staining in the Hyal1 −/− mouse femurs (Fig. 3a,b). Hyal1 −/− mice exhibit decreased femur length and decreased bone mineral density. We wondered whether the increase of bone resorption activity detected for Hyal1 −/− osteoclasts in vitro translates into an alteration of bone remodeling in vivo. Using peripheral Quantitative Computed Tomography (pQCT), we measured the bone mineral density (BMD) of right femurs isolated from 1-year-old male mice of wild-type (WT) and HYAL1 deficient (Hyal1 −/−) mice. (Table 1, n ≥ 6 per group). Three different areas of the bones located at 17.5% and 22% (metaphysis), and at 50% (diaphysis) distance from the knee joint space (distal end) were scanned. Two-tailed Mann-Whitney U statistical tests revealed a significant, ~ 25% decrease of trabecular BMD at 17.5 and 22% in the metaphysis of Hyal1 −/− femurs (p < 0.01). Total BMD was decreased by ~ 15% at these locations. A significant decrease of cortical BMD was also measured in their diaphysis (p = 0.01) but it should be noted that due to voxel size, this measurement is less accurate.
Moreover, we detected a significant decrease of the length of left femurs in Hyal1 −/− vs WT mice (with p values < 0.05; Fig. 4a). Taken together, these data point out that bone remodeling is altered in Hyal1 −/− mice and www.nature.com/scientificreports/ that this alteration leads to decreased BMD and shortened femurs. In accordance with HYAL1 affecting bone growth, histological analyses revealed a significant decrease of the epiphyseal growth plate thickness (Fig. 4b,c). In addition, a decrease of the bone area ratio relative to the total tissue area in Hyal1 −/− femurs was detected in bone sections, consistent with decreased BMD (Fig. 4c,d). Trabecular number (Tb.N) in the epiphysis was not changed, but trabecular width (Tb.Wi) was found decreased (Fig. 4e,f).
Osteoclast numbers are increased in the epiphysis and metaphysis of Hyal1 −/− mouse femurs whereas osteoblast numbers are unchanged. Osteoclasts were identified and counted in femur sections of 18-month-old WT and KO mice through the histochemical detection of TRAP (Fig. 5). This analysis revealed that the number of osteoclasts per mm, and osteoclast surface relative to bone surface (Os.S/BS), are increased in Hyal1 −/− vs WT mice both at the epiphysis ( Fig. 5a; p < 0.05 and p = 0.07, respectively). These parameters were also increased at the epiphyseal plate (Fig. 5b) though statistical threshold was only reached for osteoclast number quantifications (p < 0.05). We also analyzed the diaphysis (Fig. 5c) but found no difference in osteoclast numbers or surface at this site.
Although osteoblasts do not express HYAL1 16 , it has been reported that HA can influence the differentiation and activity of these bone forming cells in vitro 3 . Hence, we also considered that the loss of HYAL1 in osteoclasts could alter osteoblast numbers, possibly through an alteration of the catabolism of HA in Hyal1 −/− bones. To investigate this possibility, we counted osteoblasts on hematoxylin eosin saffron (HES) stained sections of WT and Hyal1 −/− femurs, and detected the osteoblast marker alkaline phosphatase by immunohistochemistry ( Fig. 6a-c). No difference was detected between mouse groups for osteoblast numbers. However, a decrease of the percentage of stained area was observed in the epiphysis (p = 0.07), suggesting a HYAL1 deficiency may inhibit osteoblast differentiation in vivo.
Circulating levels of PINP are decreased and CTX levels are increased in 18-month-old Hyal1 −/− mice. We measured the circulating levels of Procollagen type I N-terminal Propeptide (PINP), a marker of bone formation by osteoblasts, and of the osteoclast resorption marker CTX. Three groups of 18-month-old mice (with n ≥ 4 mice for each genotype in each group) were assessed independently. The results show a statistically significant decrease of PINP levels in Hyal1 −/− plasma (Fig. 7a). This finding indicates that osteoblast To do so, conditioned media were collected between day 8 and day 10 after plating and differentiation of the macrophages on bone slices (n ≥ 5 mice per group, and technical replicates were quantified). The graphs show the means ± SEM. *p < 0.05, **p < 0.01 (two-tailed Mann-Whitney U tests). www.nature.com/scientificreports/ activity is decreased, since osteoblast numbers were found unchanged on Hyal1 −/− bone sections. In addition, an increase of CTX level was detected in sera of the HYAL1 deficient groups (Fig. 7b). Coupled with the elevated CTX levels detected in the culture medium of Hyal1 −/− osteoclasts differentiated on bone slices in vitro (cf. Fig. 2h), this result points out that HYAL1 deficiency potentiates the bone resorption activity of osteoclasts.
The knock-down of Hyal1 impairs the activity of osteoblasts differentiated in vitro from mouse calvariae pre-osteoblasts. Lastly, we isolated osteoblast precursors from WT and Hyal1 −/− newborn calvariae and differentiated them into osteoblasts in vitro for 21 days. To compare the differentiation rate of the cells, we detected alkaline phosphatase activity in the wells at Day 14 using a cytochemistry assay, and measured alkaline phosphatase specific activity after cell lysis (Fig. 8a). Three independent sets (each of them derived from a pool of minimum 8 different calvariae, and with 3 replicates per set), were analyzed. No statistically relevant differences between groups were detected for the alkaline phosphatase marker (p = 0.4). However, at day 21 of differentiation, an alizarin red staining followed by quantification of optical densities in tissue extracts at 405 nm (ΔOD relative to OD measured in extracts of non-differentiated controls) highlighted decreased mineralization by Hyal1 −/− osteoblasts (Fig. 8b, p < 0.05).

Discussion
Our results demonstrate that, in addition to osteoarthritis 12 , HYAL1 deficient mice exhibit altered bone homeostasis. The shorter femurs and thinner epiphyseal growth plates in aged Hyal1 −/− mice suggest that HYAL1 plays a role in the longitudinal growth of long bones. As femur length is similarly decreased in mice deficient for another protein involved in HA depolymerization, CEMIP (previously known as KIAA1199) 17 , we can infer that undegraded HA accumulation in bone underlies these growth alterations. The major difference between the two  www.nature.com/scientificreports/  www.nature.com/scientificreports/ models is that CEMIP is highly expressed in chondrocytes (much more than in osteoclasts) and CEMIP deficient mice display specific defects in endochondral ossification, whereas HYAL1 is highly overexpressed in osteoclasts 4 .
Since HA represents at most 7% of all bone glycosaminoglycans (GAGs), with GAGs making up less than 1% of all organic bone material 2,18 , the structural contribution of HA to BMD is probably very low. This might explain that HYAL1 deficiency does not result in increased BMD, by contrast to inactivation of cathepsin K, which causes pycnodysostosis 19 . On the other hand, our results highlight that endogenous HA controls BMD indirectly, through the regulation of bone cell behavior. Hyal1 −/− mice exhibit decreased femur BMD compared to age-matched control mice. This phenotype appears accounted for, at least partly, by an increase of osteoclast numbers in the femur epiphysis and metaphysis and by an increase of osteoclast resorption activity. Little changes are detected in the diaphysis, which is not unexpected considering that cortical bone is less prone to remodeling than trabecular bone. CTX levels are elevated in Hyal1 −/− sera compared to WT mice sera, as Other groups have reported that HA influences osteoclast and osteoblast function in vitro, in a size dependent manner (reviewed in 3 ). Since the molecular mass pattern of HA molecules is not altered in other tissues of Hyal1 −/− mice 8 , it seems more likely that the increased osteoclast numbers and activity, and decreased osteoblast activity, in the absence of HYAL1, results from a general accumulation of HA rather than from a change of its molecular mass in Hyal1 −/− bones. Of note, it has been reported that HA binding to its cell surface receptor CD44 on bone marrow stromal cells increases the expression of RANKL, the cytokine that induces osteoclast differentiation 20 . HA accumulation around Hyal1 −/− osteoclasts could also explain their increased resorption activity. Indeed, it has been shown that CD44 binding to extracellular HA activates podosomes formation and maturation into actin rings, a path that is known to promote bone degradation 21,22 . Consistent with these findings, Cd44 knockout in mice results in smaller osteoclasts and shallow resorption pits 23,24 . Though actin ring numbers were found unchanged when Hyal1 −/− osteoclasts were differentiated on bone slices, the increased resorption activity of these cells may result from a longer time spent attached to the bone due to CD44-HA pairing. Regarding osteoblasts, it has been reported that HA molecules of a broad range of molecular mass can increase their proliferation, differentiation, and activity in vitro 25 . However, our results suggest that endogenous HA accumulation in bones reduces bone matrix synthesis by osteoblasts. These findings are consistent with the report of Kaneko et al., who showed that HA negatively regulates osteoblastic differentiation by binding to CD44 26 .
Up until recently, only two lysosomal hydrolases, cathepsin K and TRAP, were found overexpressed upon osteoclast differentiation. Our past research revealed HYAL1 as a third lysosomal enzyme subject to specific upregulation during the differentiation process of osteoclasts from precursor cells 4 . The data presented here add another layer of understanding by showing that HYAL1 contributes to bone remodeling. HYAL1 affects both osteoclast and osteoblast function, most likely by controlling HA concentration in bones, and a lack of HYAL1 induces osteopenia. The metabolism of HA through hyaluronidases is also involved in chondrocytes differentiation 17 and functions 27 . Hence, HA, despite its low abundance in bones, reveals itself as a fine regulator of bone structure and strength. In the future, the expression and activity of HYAL1, and the accumulation of HA, should be investigated in human bone pathological conditions, including osteoporosis and osteopetrosis, as they may represent novel pharmacological targets for medical use. were raised in our laboratory and backcrossed for 9 generations on a C57BL/6 genetic background. All experimental procedures were approved by the Animal Ethics Committee of the University of Namur and were performed in accordance with the relevant guidelines and regulations, as well as in compliance with the ARRIVE guidelines. The mice were housed at the UNamur animal facility and had free access to food and water. For one mouse group, an OVN food starvation period was included prior to blood collection for assays presented in Fig. 7. The protocols were recorded as JA14/214, JA20/356 and PM BO 21/015. Mice were anesthetized with 10 mg/kg xylazine and 100 mg/kg ketamine followed by euthanasia by cervical dislocation.
pQCT. Femurs from 1-year-old C57BL/6 male mice were cleaned of soft tissues, fixed in 10% formalin for 48 h at room temperature (RT) and stored in 70% EtOH at 4 °C. The BMD was then determined by pQCT with a XCT Research Scanner. The bones were placed in a syringe filled with 70% EtOH and BMD was measured at three different sites identified by laying out from the most distal aspect of the femoral condyles a distance corresponding to 17.5, 22 and 50% of the total length of the femur. The slices had a thickness of 0.25 mm with a voxel size of 200 µm. The CALCBD program was used to analyze the trabecular and total BMD while the CORTBD function was used to assess the cortical BMD. In order to define trabecular bone, the outer 55% of the bone cross section was concentrically excluded by the CALCBD function. Tissues with a density below 200 mg/cm 3 were not included in the analysis as they were considered as soft tissues by the software. The cortical bone parameters were determined with a threshold setting of 350 mg/cm 3 . Osteoblast differentiation in vitro. A minimum of 8 calvariae of 3-4 days old mice were used in individual preparations. Pre-osteoblasts were isolated by successive trypsin and collagenase treatments. Briefly, calvariae were first incubated for 20 min at 37 °C in Trypsin-EDTA (0.25%). Next, they were incubated for 20 min at 37 °C in DMEM/HamF12 (Gibco, Life Technologies, UK) without serum supplemented with 2% glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Lonza, Walkersville, MD, USA), as well as 3 mg/mL collagenase D (Roche diagnostics Mannheim, Germany). After discarding of the supernatant, they were re-incubated twice for 45 min in a medium with the same composition. The cells isolated in these last two steps were then pooled, counted and cultured in DMEM/HamF12 (Gibco, Life Technologies, UK) supplemented with 2% glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, and 15% fetal bovine serum (80 000 cells/well in 12-well plates Actin ring staining and bone resorption assay. Bone marrow monocytes cultured in the presence of M-CSF for 5 days were plated onto thin nasal bovine bone slices (kindly provided by Dr. Haibo Zhao, Center for Metabolic Bone Diseases, University of Arkansas for Medical Sciences, AR, USA). The differentiation into osteoclasts was then induced with RANKL as described above. 10 days later, the cells were fixed with 4% of paraformaldehyde in PBS, permeabilized with PBS-saponin 0.5% and incubated with 1% of bovine serum albumin in PBS. The cells were then incubated for 15 min at RT with 5% of Alexa 488-phalloidin in PBS (Invitrogen, Carlsbad, CA, USA) and, after several washes in PBS, for 20 min at RT with 1% of Hoechst 33258 in PBS (Molecular Probes, Eugene, OR, USA). These probes label actin and nuclei, respectively. Osteoclasts with a minimum of 3 nuclei were counted with a BX63 Olympus fluorescence microscope. Soft brushing of the surface was subsequently conducted for 10 min in PBS containing 1% of Triton X-100 and followed by an incubation of 1 min in 1 M NaOH to complete the removal of the cells. Lastly, the bone slices were washed in H 2 Od, dehydrated in EtOH, dried and observed with a JEOL 6010LV scanning electron microscope to visualize resorption pits formed by osteoclasts. For signal quantification, after imaging of the wells using an Espon scanner, the tissues were dissolved in 10% acetic acid. The pH was adjusted between 4.1 and 4.3 with NH 4 OH and the absorbance was then read at 405 nm with a Spectramax plate reader (Molecular Devices, CA, USA). ELISA assays. The following kits were used, following manufacturer's instructions: CrossLaps (CTX-I) ELISA kit, RatLaps (CTX-I) EIA kit, Rat/Mouse PINP EIA kit (Immunodiagnostic Systems, Tyne and Wear, UK).

Osteoclast differentiation in vitro.
Histology-Immunohistochemistry. Femurs from C57BL/6 male mice were fixed with 4% paraformaldehyde in PBS for 24 h at 4 °C. The bones were successively washed with PBS, PBS-glycerol 5%, PBS-glycerol 10% and PBS-glycerol 15% (12 h incubation in each solution, at 4 °C). To decalcify bones, the samples were incubated for 12 days at 4 °C using 14.5% EDTA and 15% glycerol at pH 7.3. The specimens were then washed with PBS-glycerol 15%, PBS-glycerol 10%, PBS-glycerol 5% and PBS (12 h incubation in each solution, at 4 °C). After a step of dehydration in methanol and toluol, bones were embedded in paraffin and serially cut into 6 µm sections. www.nature.com/scientificreports/ Sections were rehydrated and stained with HES. In a set of experiments, TRAP was detected as described above, followed by an hemalun staining on rehydrated bone tissue sections.
To detect HA, the sections were rehydrated, treated with 0.1 M glycine and with 3% H 2 O 2 followed by a blocking step of 1 h in PBS-BSA 0.2% containing 0.02% of Triton X-100. The slices were then incubated for 1 h at RT with a biotinylated Hyaluronic Acid Binding Protein (HABP, 1:100 dilution, Merck Millipore, Burlington, MA, USA), washed with PBS-BSA 0.2%, and incubated for 1 h at RT with streptavidin-HRP (1:50 dilution, R&D Systems, Minneapolis, MN, USA). The 3,3ʹ-diaminobenzidine technique (Liquid DAB + Substrate chromogen System, Dako, Glostrup, Denmark) was used to reveal the signals.